AB0151 DYSFERLIN AS A POTENTIAL ANTI-FIBROTIC PROTEIN INVOLVED IN TGF-Β/FOSL-2/AUTOPHAGY CROSSTALKING IN CARDIAC FIBROBLASTS UNDER IMMUNOFIBROTIC CONDITIONS
Authors: I. Kocherova, E. Pachera, D. Nurzynska, F. Di Meglio, O. Distler, P. Blyszczuk, G. Kania
Journal: Annals of the Rheumatic Diseases (2023)

<h3>Background</h3> Myocardial fibrosis, ultimately leading to heart failure (HF), represents a point of convergence for most cardiac disorders. Heart involvement is a common finding in autoimmune rheumatic patients. The TGF-β-FOSL-2-autophagy axis controls cardiac fibroblast differentiation, driving the progression of profibrotic changes [1]. Limited evidence suggests the pro-regenerative potential of adult cardiac fibroblasts directed to restrain the excessive profibrotic response and support tissue regeneration following myocardial injury. <h3>Objectives</h3> To identify the molecular basis of fibroblast-mediated compensatory response to myocardial fibrosis in adult heart. <h3>Methods</h3> Adult cardiac fibroblasts were isolated from the left atria of patients undergoing heart transplantation due to end-stage HF caused by inflammatory dilated cardiomyopathy (n=5) and from unaffected myocardium of brain-dead donors (Ctrl, n=5). Protein quantification was performed using liquid chromatography tandem-mass spectrometry (LC–MS/MS), and the data analysis was conducted with MaxQuant v1.6.2.3 software. The Enrichr tool was used for pathway enrichment analysis [2]. Selected gene knockdown was achieved through Lipofectamine 2000-mediated siRNA transfection of human foetal cardiac fibroblasts (HCFs, Sigma), untreated or stimulated with TGF-β (10 ng/mL, 48-72h). Chosen gene and protein expression was evaluated by RT-qPCR and Western Blot, respectively. Cell viability was determined with PrestoBlue HS reagent, and ATP levels were measured with CellTiter-Glo assay 48h after transfection. <h3>Results</h3> Quantitative proteomic analysis of adult cardiac fibroblasts revealed 14 differentially expressed proteins (adj. p&lt;0.05) in HF compared to Ctrl. The identified proteins belong to biological pathways mainly involved in ECM remodeling and associated with several pathologies, such as atherosclerosis, muscular dystrophy and myocardial ischemia. The most upregulated protein in HF fibroblasts was dysferlin (DYSF, log2FC=5.78, adj. p&lt;0.005), which is known to promote sarcolemma repair in skeletal muscle fibres and cardiomyocytes. Further <i>in vitro</i> study showed that TGF-β stimulation significantly upregulates DYSF gene (n=4, p&lt;0.001) and protein (n=3, p&lt;0.001) expression in cultured HCFs. <i>DYSF</i> silencing upregulated <i>COL1A1</i> expression in untreated (n=4, p&lt;0.05) and TGF-β-stimulated (48 h, n=4, p&lt;0.05) HCFs. On the protein level, <i>DYSF</i> knockdown upregulated the profibrotic transcription factor FOSL-2 in untreated HCFs (n=9, p=0.004) and following 72h of TGF-β stimulation (n=9, p&lt;0.001). In turn, <i>FOSL-2</i> silencing in HCFs significantly upregulated DYSF protein level (n=5, p&lt;0.0001) after 72 h of TGF-β stimulation, suggesting a mutual regulation between DYSF and FOSL-2. Further analysis of autophagy-related proteins revealed that <i>DYSF</i> silencing increases LC3BII expression (n=6, p&lt;0.05) and downregulates P62 (n=6, p=0.01) in TGF-β-stimulated HCFs. Finally, we observed reduced viability of cells subjected to <i>DYSF</i> knockdown in both untreated (n=4, p&lt;0.01) and TGF-β-stimulated (n=4, p&lt;0.05) conditions. <i>DYSF</i> silencing was also associated with decreased ATP production in TGF-β-treated HCFs (n=6, p=0.05). <h3>Conclusion</h3> Proteomic profiling of human cardiac fibroblasts supported by <i>in vitro</i> analysis identified DYSF as an anti-fibrotic protein compensatory upregulated under immunofibrotic conditions and involved in TGF-β-FOSL-2-autophagy axis regulation. This newly proposed candidate may serve as a potential therapeutic target for stimulating fibroblast-induced myocardial regeneration. <h3>References</h3> [1]Seidenberg, J.; Stellato, M.; Hukara, A.; Ludewig, B.; Klingel, K.; Distler, O.; Błyszczuk, P.; Kania, G. The AP-1 Transcription Factor Fosl-2 Regulates Autophagy in Cardiac Fibroblasts during Myocardial Fibrogenesis. Int. J. Mol. Sci. 2021, 22, 1–19. [2]Enrichr Available online: https://maayanlab.cloud/Enrichr/# (accessed on Dec 16, 2022). <h3>Acknowledgements:</h3> NIL. <h3>Disclosure of Interests</h3> Ievgeniia Kocherova: None declared, Elena Pachera: None declared, Daria Nurzynska: None declared, Franca Di Meglio: None declared, Oliver Distler Speakers bureau: Bayer, Boehringer Ingelheim, Janssen, Medscape, Consultant of: 4P-Pharma, Abbvie, Acceleron, Alcimed, Altavant Siences, Amgen, AnaMar, Arxx, AstraZeneca, Baecon, Blade, Bayer, Boehringer Ingelheim, Corbus, CSL Behring, Galapagos, Glenmark, Horizon, Inventiva, Kymera, Lupin, Miltenyi Biotec, Mitsubishi Tanabe, MSD, Novartis, Prometheus, Redxpharna, Roivant, Sanofi and Topadur, Grant/research support from: Kymera, Mitsubishi Tanabe, Boehringer Ingelheim, Przemyslaw Blyszczuk: None declared, Gabriela Kania: None declared.